The goal of this project is to develop an integrated diagnostic system designed for rapid isolation, detection, and multiplexed identification of circulating exosomal microRNA (miRNA) biomarkers in human clinical samples for early detection of diseases. Dysregulation of miRNAs is often observed in various diseases including cancer, cardiovascular illnesses, and infection. Recent studies have also demonstrated that miRNAs can be secreted from tumor cells into bloodstream via exosomes; their expression profiles can be used to classify cancer types. Therefore, circulating exosomal miRNAs are considered promising biomarkers for early detection and diagnosis of cancer. However, because of technical difficulties arising from exosome isolation and miRNA analysis, detection of these small molecules has not been adopted into clinical practice. Thus, there is a need to develop alternative analysis strategies that could offer more advantages over conventional methods. In this project, we propose an integrated acoustofluidic plasmonic molecular diagnostic system designed for rapid isolation and multiplexed detection of exosomal miRNA biomarkers. The cross-disciplinary approach will provide new capabilities to advance the precise clinical diagnosis of diseases. This system integrates two unique technologies: (1) acoustofluidic technology that can rapidly isolate exosomes with high yield and purity; and (2) surface-enhanced Raman scattering (SERS)-based ?Inverse Molecular Sentinel? (iMS) nanoprobes for direct miRNA detection. Although miRNAs related to colorectal cancer (CRC) will be used as the model system, the proposed project will also lead to the development of a generally applicable point-of-care diagnostic technology for other types of diseases. The specific aims are: (1) Develop and integrate an acoustofluidic system for exosomal miRNA isolation; (2) Develop iMS nanoprobes for multiplexed detection of exosomal miRNA biomarkers; and (3) Technical Evaluation of the SERS-acoustofluidic system. We will build upon the combined knowledge of our interdisciplinary team establishing the technical validation required to ready this integrated technology for future use in clinical settings. Throughout this project, an integrated system will be developed and validated for in-situ analysis of multiple exosomal miRNAs from clinical samples without the need for miRNA extraction and amplification. In the technical validation phase, the results will be compared to those obtained using conventional assays (e.g. qRT-PCR). The system will lead to a no-sample preparation and ?sample-to- answer? analysis approach that is based on the integration of existing and tested sub-components. This new diagnostic system is capable of enhancing research and translation in the areas of early detection and screening of various diseases beyond CRC; it is exceedingly well suited for point-of-care testing. The proposed system represents a major innovation with transformative potential in biomedical research, diagnostics, and screening.